ES2347566T3 - IMMUNOMODULATOR THAT INCLUDES WHOLE CELLS OF BACTERIA TSUKAMURELLA. - Google Patents

Abstract

Use of an immunomodulator composition or a pharmaceutical composition comprising an entire cell of a bacterium of the genus T. sukamurella in the manufacture of a medicament for the treatment or prevention of an autoimmune disease or autoimmune disorder.

Description

FIELD OF THE INVENTION

The present invention relates to immunomodulators, in particular vaccines that modulate a cellular immune response and its uses. The present invention relates to the use of immunomodulators in the manufacture of a medicament for the treatment and / or prevention of an autoimmune disease or autoimmune disorder, including certain vascular disorders.

BACKGROUND OF THE INVENTION

The immune system is a reticulum of complicated cells and cellular components (molecules) that normally work to defend the body and eliminate infections caused by bacteria, viruses, and other foreign invading bodies. If a person has an autoimmune disease, the immune system attacks itself by mistake, targeting the cells, tissues and organs of the person's own body. A collection of cells and molecules of the immune system at a target site is called inflammation.

There are many different autoimmune diseases, and each of them can affect the body in different ways. Many of the autoimmune diseases are rare. As a group, however, autoimmune diseases affect millions of people.

Some autoimmune diseases are known to begin or worsen with certain triggers such as viral, parasitic and chronic bacterial infections. Other less understood influences affect the immune system and the course of autoimmune diseases include aging, chronic stress, hormones and pregnancy.

Autoimmune diseases are often chronic, requiring care and control throughout life, even when the person seems well or feels well. Currently, few autoimmune diseases can be cured or caused to go into remission with treatment.

Most doctors often help patients treat the consequences of inflammation caused by autoimmune disease. In some people, a limited number of immunosuppressive medications may result in disease remission. However, even if their disease is in remission, patients are rarely able to modify their medication. The long-term side effects of immuno-suppressive medication can be substantial.

The onset and progression of vascular damage is a complex, multi-factorial process, but there is growing evidence that inflammatory responses play a key role. Vascular damage is involved in the development of atherosclerosis, and in thrombotic processes that lead to acute ischemic syndromes such as myocardial infarction, stroke and peripheral arterial occlusion.

Immune mechanisms may be important in the development and maintenance of atherosclerosis and myointimal hyperplasia (MIH).

Myointimal hyperplasia (MIH) can be considered as an exaggerated healing response to damage such as balloon angioplasty. A cascade of episodes results in: loss of the basement membrane, migration of vascular smooth muscle cells

(VSMC) from the middle in the intima, VSMC proliferation and phenotypic change to a more secretory fibroblast cell type and increased extracellular matrix production, which eventually leads to stenosis or vessel occlusion. It occurs after bypass grafting and balloon angioplasty and affects approximately 30% of such cases in clinical practice. It is the main cause of failure of such procedures and treatment of the vessels / grafts with stenosis and resulting blockages is problematic. The mechanisms of underlying cells that lead to MIH are not well understood and to date no therapy has been developed that can effectively prevent it. The clinical relevance of the current patent has to do with the large number of coronary artery angioplasties performed annually in the United Kingdom and in the world. Although permanent vascular dilators that elute the drug are currently being produced, the results are unlikely to completely avoid restenosis. Any safe, relatively inexpensive complementary therapy, such as the immunotherapy proposed in this patent, would have a significant clinical impact.

The mechanisms involved in immunotherapy against restenosis are complex and not completely clarified. Endothelial damage caused by angioplasty can be exacerbated by the host's immune response to hsp. Hsp are proteins produced by stressed cells that have been implicated in the pathogenesis and pathophysiology of various immune disorders including atherosclerosis (Xu Q et al. Arterioscler Thromb 1992; 12: 789-799). They are likely to be present in endothelial and smooth muscle cells in the region of an angioplasty. In effect, hsp acts as an autoantigen that can then be attacked by the immune system. This situation can be induced experimentally by immunization with a mycobacterial cross-reaction hsp (hsp65) that leads to endothelial lesion in rabbits and mice (Xu Q, et al. Arterioscler Thromb 1992; 12: 789-799 and George J , et al. Circ. Res. 2000; 86: 1203-1210). The effect appears to be IL-4 dependent secreted by Th2 lymphocytes, and is probably mediated by antibody George J, et al. Circ. Res. 2000; 86: 1203-1210 and Schett G, et al. J. Clin. Invest. nineteen ninety five; 96: 2569-2577). The relevance of these observations to man is suggested by the ability of the human affinity purified antibody eluted from hsp65 columns to damage human endothelial cells stressed in vitro. This finding suggests that the antibody cross-reacts with hsp60 which is the human homologue of hsp65, and may be accessible to the antibody when expressed in the membranes of stressed endothelial cells. It has been suggested that such antibodies that bind to stressed endothelial cells may be a factor in the production of coronary artery disease after heart transplantation (Crisp SJ et al. J Heart Lung Transplant 1994; 81 91). Mukherjee et al (Thromb Haemost 1996; 75: 258-60) showed no association between preoperative antibody levels to hsp65 and coronary restenosis, but showed that those patients in whom the levels of such antibodies fell after angioplasty were less likely to suffer from restenosis. In fact, the role of antibodies to hsp can be complex, because patients with vascular disease have not only increased levels of antibodies, but also their own hsp (Wright BH, et al Heart Vessels 2000; 15: 18-22 ). Thus an obvious drop in antibody levels may merely reflect an increase in protein levels. In addition, hsp have regulatory effects, and bind to arterial smooth muscle cells, which lead to an increase in survival without a requirement for internalization (Johnson AD et al. Atherosclerosis 1990; 84: 111-119).

WO 03/049752 describes compositions comprising components prepared from gram positive bacteria for the prevention and / or treatment of disorders comprising an immune dysregulation such as cancer, autoimmune diseases, allergy and tuberculosis.

SUMMARY OF THE INVENTION

The present invention is based on the surprising finding that an entire cell of a bacterium of the genus Tsukamurella administered to a test subject can induce a modification of the immune system, in particular the cellular immune system of that test subject, which effects a preventive effect. and / or therapeutic on autoimmune diseases or autoimmune disorders, particularly those that involve inflammation of the intima of blood vessels for example.

An advantage of the use of compositions comprising a whole cell of a bacterium of the genus Tsukamurella to effectively treat and / or prevent autoimmune diseases and autoimmune disorders, particularly those that involve inflammation of the intima of blood vessels for example, may be that that treatment and / or prevention is carried out while producing less long-term side effects than chemotherapies,

that is, immunosuppressive medication, now used routinely.

The phrase "cellular immune system," as used herein, includes a cell-mediated immune response that depends on the presence of T lymphocytes. The term "T lymphocytes" includes cytotoxic T lymphocytes, helper T cells, suppressor T cells. and regulatory T cells. Modification of a cell-mediated immune response can be used, for example, to overcome cell-mediated immune disorders that include, for example, autoimmune diseases or disorders.

The terms "modular", "modify", "modification" and other derivatives thereof, as used herein, mean regulation by decrease, inhibition, induction, stimulation, regulation by increment, alteration or otherwise affecting to a component or components of the cellular immune system.

DETAILED ASPECTS OF THE PRESENT INVENTION

The present invention further provides the use of an immunomodulator composition or a pharmaceutical composition comprising an entire cell of a bacterium of the Tsukamurella genus, in the manufacture of a medicament for the treatment or prevention of an autoimmune disease or an autoimmune disorder.

Suitably, the autoimmune disease or autoimmune disorder is of the type where the subjects possess damage to the immune system of one or more of the subject tissues. Suitably, the autoimmune response can be triggered by something in the subject or something in the subject's environment.

Suitably the autoimmune disease or autoimmune disorder according to the present invention may be one that follows the cause of onset. For example, the autoimmune disease or autoimmune disorder according to the present invention may be one that is caused by infection and / or some other cause of onset. Potential onset causes can be, for example, advanced age, infection (for example, parasitic infection), steroid treatment, repeated vaccination with alum, pregnancy and / or cancers.

The present invention further provides the use of an immunomodulator composition or a pharmaceutical composition comprising an entire cell of a bacterium of the genus Tsukamurella, in the manufacture of a medicament for the treatment or prevention of an autoimmune disease, where the autoimmune disease or disorder Autoimmune involves inflammation of the intima of a blood vessel.

Suitably, the autoimmune disease or autoimmune disorder according to the present invention may, in addition, involve inflammation of the intima of a blood vessel, involve inflammation of the muscular layer of a blood vessel or myocardium.

Suitably, the autoimmune disease or autoimmune disorder according to the present invention may be one that is preceded or caused by a vascular disorder.

The autoimmune disease or autoimmune disorder according to the present invention may be one or more of the following: arthritis, particularly rheumatoid arthritis, psoriasis, psoriatic arthropathy, scleroderma, thyroiditis, intimal hyperplasia after transplantation, graft rejection and vascular disorders

Suitably, vascular disorders according to the present invention include any disease or vascular disorder comprising an autoimmune element, for example one that is caused by an autoimmune response.

Suitably, vascular disorders according to the present invention may include one or more of Raynaud's disease and phenomenon, anterior uveitis, obliterating vascular disorder, atheroma formation (also known as arteriosclerosis), arteritis, myointimal hyperplasia (natural or after angioplasty), thickening, inflammatory and autoimmune of the intimate and / or muscular layer of blood vessels, inflammatory lesions of the blood vessels, atherosclerotic heart disease, reperfusion injury, cardiac conduction abnormalities, myocarditis, myocardial infarction.

Suitably, the rejection according to the present invention may be chronic graft rejection, particularly in the absence of an immunosuppressant. Thus, the composition according to the present invention can be used as a replacement for the conventional immunosuppressant administered before, during and / or after transplantation. Compositions according to the present invention can be used when transplanting cells, tissues and natural or artificial organs, such as one or more of the following: corneas, bone marrow, organs (eg, kidney, liver), crystalline, pacemakers , natural or artificial skin tissue, islet cells.

The present invention further provides the use of an immunomodulator composition or a pharmaceutical composition comprising an entire cell of a bacterium of the Tsukamurella genus, in the manufacture of a medicament for the treatment or prevention of a vascular disorder.

The present invention also provides the use of an immunomodulator composition or a pharmaceutical composition comprising an entire cell of a bacterium of the genus Tsukamurella, in the manufacture of a medicament for the treatment or prevention of arthritis, in particular rheumatoid arthritis.

The present invention further provides the use of a composition of

immunomodulator or a pharmaceutical composition comprising an entire cell of a bacterium of the genus Tsukamurella, in the manufacture of a medicament for the treatment or prevention of graft rejection.

In a further aspect the present invention provides the use of an immunomodulator composition or a pharmaceutical composition comprising an entire cell of a bacterium of the genus Tsukamurella, in the manufacture of a medicament for the treatment

or prevention of psoriasis. The term "immunomodulator", as used herein, means a substance that modulates a cellular immune system of a subject.

The term "whole cell," as used herein, means a bacterium that is intact, or substantially intact. In particular, the term "intact" as used herein means a bacterium comprising all components present in an entire cell, in particular a whole, viable cell, and / or a bacterium that has not been treated in a manner specific to remove one or more components from it. By the term "substantially intact" as used herein, it means that although the entire isolation and / or purification process used in obtaining the bacteria can result in, for example, a slight modification to the cell and / or in the elimination of one or more of the components of the cell, the degree to which such modification and / or elimination occurs is negligible. In particular, a substantially intact cell according to the present invention has not been specifically treated to remove one or more components therefrom.

Although it has been suggested that the individual components of bacterial cells could be used to induce an adjuvant effect, prior to the present invention the use of whole cells of the bacterium of the genus Tsukamurella according to the present invention was not contemplated. Surprisingly, it has been found that through the use of an entire cell of a bacterium of said genus, treatment and / or prevention of an autoimmune disease or an autoimmune disorder can be carried out. The modulation of a cellular immune response caused by the administration of said whole cell of said bacterium can be advantageously of long duration when compared with the response caused by the administration of an individual component of the bacterium.

Preferably, the composition according to the present invention comprises more than one whole cell, and more preferably comprises a plurality of whole cells.

Suitably, the immunomodulator composition according to the present invention may comprise an antigen and an adjuvant, wherein said adjuvant comprises an entire cell of a bacterium of the Tsukamurella genus. Such antigens may be those shared by these genera with other microorganisms and eukaryotic mitochondria, for example vertebrate cells.

In another aspect, the immunomodulator composition may be a pharmaceutical composition comprising an entire cell of a bacterium of the Tsukamurella genus and optionally a pharmaceutically acceptable carrier, diluent or excipient, said immunomodulator composition in use modifies a cellular immune response.

In a further aspect, the immunomodulator composition and / or pharmaceutical composition may comprise an entire cell of a bacterium of the genus Tsukamurella, and at least one added cytokine, such as interleukin 2 for example. Cytokine can help strengthen the immune modulating action of the present invention.

Suitably, the antigen or antigenic determinant may be an antigen or antigenic determinant of one or more of the following: BCG vaccine (Bacillus Calmette and Guerin), diphtheria toxoid vaccine, diphtheria / tetanus / pertussis vaccine (DTP or Triple), pertussis vaccine, tetanus toxoid vaccine, measles vaccine, mumps vaccine, rubella vaccine, OPV vaccine (oral polio vaccine) and Mycobacteria vaccae, or part thereof (as taught in document GB0025694.1). This list is by no means limiting and suitable antigens from other sources can be added to the compositions according to the present invention so that responses to those antigens can also benefit from the induced regulation of the response that is produced from the administration of the composition according to the present invention. Other suitable antigens may be antigens of other viruses, tumors, parasites or other bacteria of unnatural origin present in the modulator composition according to the present invention.

Suitably, the immunomodulator composition and / or pharmaceutical composition may comprise two or more such antigens or antigenic determinants.

In a further aspect, the immunomodulator composition or pharmaceutical composition comprising an entire cell of a bacterium of the Tsukamurella genus according to the present invention can be used as a vaccine.

Suitably, the vaccine can be a prophylactic vaccine or a therapeutic vaccine.

In a further aspect, the present invention provides an immunomodulator composition or a pharmaceutical composition comprising an entire cell of a bacterium of the genus Tsukamurella for use in the treatment or prevention of an autoimmune disease or an autoimmune disorder.

In one aspect, the entire cell of the bacterium according to the present invention

You can regulate by decrease a Th2 response.

In another aspect, the entire bacterium cell according to the present invention can regulate by increasing a Th1 response.

Suitably, the entire cell of the bacterium according to the present invention can regulate by decreasing a Th2 response and regulating by increasing a Th1 response.

Alternatively, the entire cell of the bacterium according to the present invention can regulate by increasing a Th1 response while not affecting a Th2 response.

Alternatively, the entire cell of the bacterium according to the present invention can regulate by decreasing a Th2 response, while also regulating by increasing a Th1 response.

Alternatively, the entire cell of the bacterium according to the present invention can regulate by increasing a Th2 response, while also regulating by increasing a Th1 response.

A method for treating or preventing an autoimmune disease or an autoimmune disorder that comprises administering an effective amount of a pharmaceutical composition and / or immunomodulator composition comprising an entire cell of a bacterium of one or more of the genera Rhodococcus, Gordonia, Nocardia, Dietzia, Tsukamurella and Nocardioides to a subject in which said composition modulates a cellular immune response is also taught herein.

Suitably the effective amount of the pharmaceutical composition and / or immunomodulator composition can be administered in the form of an individual dose. Alternatively, the effective amount of the pharmaceutical composition and / or immunomodulator composition may be administered in multiple (repeated) doses, for example two or more, three or more, four or more, five or more, ten or more, or twenty or more repeated doses.

In particular, such repeated doses may be necessary for the treatment of established or chronic conditions, for example.

A method for protection, which includes immunization, a subject against an autoimmune disease or an autoimmune disorder comprising the administration of a pharmaceutical composition and / or immunomodulator composition comprising an entire cell of a bacterium of the genera Rhodococcus, Gordonia, Nocardia, Dietzia, Tsukamurella and Nocardioides are taught in this document.

The term "protected" as used herein means that the subject is less susceptible to the disease / disorder when compared to an untreated or administered subject with the compositions according to the present invention and / or that the subject is more capable of counteracting or overcoming the disease / disorder when compared to an untreated or administered subject with the compositions according to the present invention.

An effective amount of a pharmaceutical composition and / or an immunomodulator composition according to the present invention can be administered to a subject, wherein said composition is co-administered with an antigen or antigenic determinant.

When the composition is co-administered with an antigen or antigenic determinant according to the present invention, the antigen or antigenic determinant may suitably be an antigen or antigenic determinant of one or more of the following: BCG (Calmette bacillus vaccine) and Guerin), diphtheria toxoid vaccine, diphtheria / tetanus / pertussis vaccine (DTP or Triple), whooping cough vaccine, tetanus toxoid vaccine, measles vaccine, mumps vaccine, rubella vaccine, OPV (vaccine oral polio) and Mycobacteria vaccae, or part thereof (as taught in GB0025694.1). Suitably two or more, or three or more, of such antigens or antigenic determinants may be co-administered with a pharmaceutical composition or an immunomodulator composition according to the present invention.

Preferably, a medicament according to the present invention is used for the treatment or prevention of an autoimmune disease or an autoimmune disorder.

In a further aspect of the present invention, the pharmaceutical composition or the immunomodulator composition according to the present invention may comprise bacteria of the genus Tsukamurella. Suitably, the composition may comprise two or more, or three or more, bacteria of any of the Rhodococcus, Gordonia, Nocardia, Dietzia, Tsukamurella and Nocardioides genera, being at least one Tsukamurella bacteria.

Preferably, the bacteria for use according to the present invention are of the genus Tsukamurella, including any species of this genus such as Tsukamurella paurometabola and Tsukamurella inchonensis for example. Suitably, the species used are those that can be grown on medium, which is a low antigenic medium, preferably non-antigenic. By way of example only, a suitable non-antigenic medium is Sauton medium.

Preferably, the bacterium according to the present invention is destroyed before use. Suitably, the bacterium according to the present invention can be destroyed by heat treatment thereof, for example, heat treatment in a

autoclave at 121 ° C for 15 minutes.

Other suitable treatments to destroy the bacteria may include ultraviolet or ionizing radiation or treatment with chemical compounds such as phenol, alcohol or formalin.

Suitably, the bacteria according to the present invention can be purified and / or isolated.

Suitably, the bacterium according to the present invention can be suspended in water or buffered saline, suitably buffered borate at pH

8.

The term "subject", as used herein, means an animal. Suitably, the subject can be for example any animal, including birds, crustaceans (such as shrimp for example), fish and mammals. Preferably, the subject is a mammal, including for example cattle and humans. In some aspects of the present invention, the subject may suitably be a human being.

When the pharmaceutical composition or immunomodulator composition is administered (for the first time if more than one administration is to be performed) to cattle, it is preferably administered after the cattle have suckled the first time. In particular, for some applications it may be important to allow the baby to take and / or digest the paternal colostrum before the administration of the first (when done more than once) or only the dose of the pharmaceutical composition or immunomodulator composition. For the avoidance of doubt, for some applications the first administration of the pharmaceutical composition or immunomodulator composition should be provided between about 1-4 days after birth, preferably 1-3 days after birth, more preferably 1-2 days after birth, preferably 2-3 days after birth. Then administrations can be provided 7 days and / or 8 12 weeks after the first injection.

The term "immunomodulator" as used herein includes a vaccine.

THERAPEUTIC USES

The immunomodulators of the present invention can be used in therapy. In particular, such compounds can be used to modulate T lymphocyte responses in vivo and / or other cells involved in an immune response in vivo.

Immunomodulator / pharmaceutical compositions capable of modulating, in particular blocking, T cell proliferation and / or differentiation and / or activity can be used against any disorder that is susceptible to prevention or treatment by modulating an adaptive immune response, that is, a cellular immune response.

Suitably, the autoimmune disease or autoimmune disorder according to the present invention is of the type in which the subjects possess damage to the immune system of one or more of the subject tissues. Suitably, the autoimmune response can be triggered by something within the subject or something within the subject's environment.

Suitably the autoimmune disease or autoimmune disorder according to the present invention may be one of those that follows a cause of onset. For example, the autoimmune disease or autoimmune disorder according to the present invention may be one that is caused by infection and / or some other cause of onset. Potential onset causes can be by way of example, old age, infection (eg parasitic infection), steroid treatment, repeated vaccination with alum, pregnancy and /

or cancers

Suitably, the autoimmune disease or autoimmune disorder according to the present invention may be one that involves inflammation of the intima of a blood vessel.

Suitably, the autoimmune disease or autoimmune disorder according to the present invention may also involve inflammation of the intima of a blood vessel, imply inflammation of the muscular layer of a blood vessel or myocardium.

Suitably, the autoimmune disease or autoimmune disorder according to the present invention may be one that is preceded or caused by a vascular disorder.

The autoimmune disease or autoimmune disorder according to the present invention may be one or more of the following: arthritis, particularly rheumatoid arthritis, psoriasis, psoriatic arthropathy, scleroderma, thyroiditis, intimal hyperplasia after transplantation, graft rejection and vascular disorders

Suitably, vascular disorders according to the present invention may include disease or vascular disorder comprising an autoimmune element, for example one that is caused by an autoimmune response.

Suitably, vascular disorders according to the present invention may include one or more of Raynaud's disease and phenomenon, anterior uveitis, obliterating vascular disorder, atheroma formation (sometimes known as arteriosclerosis), arteritis, myointimal hyperplasia (natural or after angioplasty), inflammatory and autoimmune thickening of the intima and / or muscular layer of the blood vessels, inflammatory lesions of the blood vessels, atherosclerotic heart disease, lesion of

reperfusion, cardiac conduction abnormalities, myocarditis, myocardial infarction.

Suitably, graft rejection according to the present invention may be chronic graft rejection, particularly in the absence of an immunosuppressant. Thus, the composition according to the present invention can be used as a replacement for the conventional immunosuppressant administered before, during and / or after transplantation. Compositions according to the present invention can be used when transplanting cells, tissues and natural or artificial organs, such as one or more of the following: corneas, bone marrow, organs (eg, kidney, liver), crystalline, pacemakers , natural or artificial skin tissue, islet cells.

AUXILIARY CELLS T

The term 'Th1' as used herein refers to a type 1 of auxiliary cell T (Th1). The term can also be used herein to refer to the response mediated by or by such a cell type. Such response may include one or more of the secretion of interleukin-2 (IL-2), Interferon-gamma secretion (IFN-y), macrophage activation, activation of cytotoxic T cells, or any other episode associated with Th1. Thus, the term 'Th1' can include Th1 cell (s) as well as the immune response (s) that produces such cell (s).

The term 'Th2' as used herein refers to a type 2 of auxiliary cell T (Th2). The term can also be used herein to refer to the response mediated by or through such a cell type. Such a response may include one or more of the secretion of Interleukin-4 (IL-4), the secretion of interleukin IL-402 from ayuste variant, the secretion of Interleukin-5 (IL-5), increase in the levels of determinant of 30 cells (CD30) in lymphocytes, increased levels of Immunoglobulin-E (IgE) in the blood or eosinophils in the blood, or any other episode associated with Th2. Thus, the term 'Th2' can include Th2 cell (s) as well as the immune response (s) that such a cell (s) produces (n).

It is known that various conditions may result or occur from an unregulated or improperly regulated cellular immune response, in particular in the activation and / or proliferation of Th1 and / or Th2, which if it is unregulated or regulated from Inappropriate manner has been found to result in one or more detrimental effects on the subject.

An unregulated or improperly regulated cellular immune response has also been observed in autoimmune disorders such as for example inflammatory vascular diseases such as arteriosclerosis, myointimal hyperplasia after angioplasty and anterior uveitis, and during graft transplantation / rejection. As an additional example, Stansby et al. Eur 25 J Vasc Endovasc Surg 2002; 23: 23-28 tested the hypothesis that treatment with a mycobacterial preparation that modulates the antibody response should decrease vascular disease, that is, restenosis in a rat angioplasty model. It has been shown that immunomodulation with mycobacterial material suitable for use in humans can reduce MIH. Since such modulation has a low risk, this increases the hope of an important new therapeutic modality to combat restenosis.

In accordance with the foregoing, an objective of the present invention is to promote and establish the regulation of a cellular immune response, including the regulation or modulation of Th1 and / or Th2, so as to overcome the negative effects of the non-cellular immune response. improperly regulated or regulated.

Suitably, the use of an immunomodulator composition and / or pharmaceutical composition according to the present invention modulates the Th1 or Th2 response, that is, a Th1 or Th2 response that results, for example, tissue damage. .

Suitably, the use of an immunomodulator composition and / or pharmaceutical composition according to the present invention can reduce the Th1 response and reduce the Th2 response.

Suitably, the use of an immunomodulator composition and / or pharmaceutical composition according to the present invention can increase the Th1 response without affecting the Th2 response.

Suitably, the use of an immunomodulator composition and / or pharmaceutical composition according to the present invention can increase the Th1 response and decrease the Th2 response.

Suitably, the use of an immunomodulator composition and / or pharmaceutical composition according to the present invention can increase the Th1 response and increase the Th2 response.

Suitably, those skilled in the art can test specific species of each genus according to the present invention to determine their specific Th1 / Th2 response.

An unregulated or improperly regulated immune response may play a role in establishing a disease due to the fact that some diseases cause, or are a consequence of, displaced Th1 and / or Th2 responses. Accompanying these atypical reactions of Th1 and Th2 are a series of abnormal inflammatory responses, which may take part in the mechanisms underlying the

tissue pathology.

By way of example only, the immunomodulator composition and / or pharmaceutical composition according to the present invention can counteract an autoimmune disease or autoimmune disorder.

VACCINES

The preparation of vaccines containing one or more substances as an active ingredient (s), is known to those skilled in the art. Typically, such vaccines are prepared as injectables, or as liquid solutions or suspensions; Solid forms suitable for solution in, or suspension in, liquid before injection can also be prepared. The preparation can also be emulsified, or the ingredient (s) encapsulated in liposomes. The active ingredients are often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. Alternatively, the vaccine can be prepared, for example, to be ingested orally and / or capable of inhalation.

In addition, if desired, the vaccine may contain smaller amounts of auxiliary substances such as wetting or emulsifying agents and pH buffering agents.

ADMINISTRATION

Typically, a physician will determine the actual dose of a vaccine, immunomodulator composition and pharmaceutical composition that will be the most appropriate for the individual subject and will vary with the age, weight and response of the particular patient. The lower dosages are exemplary of the average case. Of course, they can be individual cases where higher or lower dosages are worthwhile

Preferably, the actual dose that is used results in minimal toxicity to the subject.

The compositions of the present invention can be administered by direct injection. The composition may be formulated for parenteral, mucosal, intramuscular, intravenous, subcutaneous, intraocular, intradermal or transdermal administration.

Suitably, the composition according to the present invention can be administered at a dose of 1 nanogram to 100 milligrams of organisms, preferably 10 nanograms to 10 milligrams of organisms, more preferably 100 nanograms to 5 milligrams of organisms, and even more preferably 100 nanograms to 1 milligram of organisms. Typically, the composition according to the present invention can be administered at a dose of 100 micrograms to 1 milligram of bacteria for human and animal use.

If the compositions of the present invention are to be administered as immunopotentiators, then 1 nanogram to 100 milligrams of organisms per dose, preferably 10 nanograms to 10 milligrams of organisms per dose, more preferably 100 nanograms to 5 milligrams organisms per dose, and even more preferably 100 nanograms at 1 milligram organisms per dose, and even more preferably, 100 micrograms at 1 milligram bacteria per dose for human and animal use can be administered at regular intervals.

As those skilled in the art will readily appreciate, the dosage administered will depend on the organism to which the dose is administered.

The term "administered" includes administration by administration mechanisms including injection, lipid-mediated transfection, liposomes, immunoliposomes, lipofectin, cationic facial amphiphiles (CFA) and combinations thereof, or even viral administration. The routes of such mechanisms of administration include but are not limited to the mucosal, nasal, oral, parenteral, gastrointestinal, topical, or sublingual routes.

The term "administered" includes but is not limited to mucosal administration, for example, in the form of a nasal spray or aerosol for inhalation or in the form of a solution that can be ingested, capsule or tablet; a parental route where administration is by an injectable form, such as, for example, an intravenous, intramuscular, intradermal or subcutaneous route.

The term "co-administered" means that the site and time of administration of each adjuvant (s), antigen (s) and / or antigenic determinant (s) of the present invention are such that the necessary modulation of the immune system. Thus, while the antigen (s) and adjuvant (s) can be administered at the same time and in the same place, it can therefore be advantageous in the administration of the antigen ( s) and / or antigenic determinant (s) at a different time and at a different place the adjuvant (s). the antigen (s) and / or antigenic determinant (s) and adjuvant (s) can even be administered in the same administration vehicle - and the antigen (s) and / or determinant (s) ) antigen (s) and adjuvant (s) may be coupled and / or not coupled and / or genetically coupled and / or uncoupled. By way of example only, the immunomodulator composition according to the present invention can be administered before, at the same time or after the administration of one or more additional antigens or antigens.

The antigen, antigenic determinant, peptide or homologue or mimetic thereof can be administered separately or co-administered to the host subject as a single dose or in multiple doses.

The immunomodulator composition and / or pharmaceutical composition of the invention can be administered by a number of different routes such as injection (including parenteral, subcutaneous, intradermal and intramuscular injection) intranasal, mucosal, oral, intra-vaginal, urethral or ocular administration .

Preferably, in the present invention, administration is by injection. More preferably the injection is intradermal.

Preferably, in the present invention, administration is by an orally acceptable composition.

For vaccination the composition may be provided in 0.1 to 0.2 ml of aqueous solution, preferably buffered physiological saline, and administered parenterally, for example by intradermal inoculation. The vaccine according to the invention is preferably injected intradermally. Slight swelling and redness sometimes itching can also be found at the injection site. The mode of administration, the dose and the number of administrations can be optimized by those skilled in the art in a known manner.

ANTIGENS US

As used herein, an "antigen" means an entity that, when introduced into an immunocompetent host, modifies the production of a specific antibody or antibodies (s) that can be combined with the entity, and / or modifies the relevant Th response, such as Th2 and / or Th 1. The antigen can be a pure substance, a mixture of substances or soluble or particulate material (including cells or cell fragments or sonic cell). In this sense, the term includes any suitable antigenic determinant, cross-reaction antigen, alloantigen, xenoantigen, allergen, allergen, hapten, and immunogen, or parts thereof, as well as any combination thereof, and these terms are used interchangeably. throughout the entire text.

The term "antigenic determinant or epitope" as used herein refers to a site on an antigen that is recognized by an antibody or T cell receptor, or is responsible for eliciting the T helper cell response. Preferably it is a short peptide derived from or part of a protein antigen. However, the term is also intended to include glycopeptides and carbohydrate epitopes. The term also includes modified amino acid or carbohydrate sequences that stimulate responses that recognize the entire organism.

A "preventive" or "prophylactic" vaccine is a vaccine that is given to unexposed individuals to prevent the development of a condition, such as by stimulating protective immunity.

A "therapeutic" vaccine is a vaccine that is given to individuals with an existing condition to reduce or minimize the condition or eliminate the immunopathological consequences of the condition.

Adjuvants

The term 'adjuvant' as used herein means an entity capable of increasing or participating in the influence of an immune response. An adjuvant is any substance or mixture of substances that helps, increases, regulates down, modifies or diversifies the immune response to an antigen.

The immunomodulator composition and / or pharmaceutical composition according to the present invention may comprise one or more adjuvants that enhance the efficacy of the immunomodulator composition and / or pharmaceutical compositions. Examples of additional adjuvants that may be effective include but are not limited to: aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate (alum), beryllium sulfate, silica, kaolin, carbon, water-in-oil emulsions, and oil in water, muramyl dipeptide, bacterial endotoxin, lipid X, Corynebacteria parvum (Propionobacteria acnes), Bordetella pertussis, Mycobacteria vaccae, polypyribonucleotides, sodium alginate, lanolin, Iisolecithin, vitamin A, interleukins such as interleukin 2, interleukin 2, saponin l12, interleukin 2, saponin 12, interleukin 2, saponin 12, interleukin 2, saponin 2, interleukin 2, sapona lomas 2, interleukin 2, sapona lomas 12 Levamisole, DEAE-dextran, blocked copolymers or other synthetic adjuvants. Such adjuvants are commercially available from various sources, for example, Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.) or Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Michigan). Only aluminum hydroxide is approved for human use. Some of the other adjuvants, such as M. vaccae for example, have been approved for clinical trials.

Suitably, the adjuvant can be an entire cell of a bacterium of any one of the genus Tsukamurella.

In the art, it is known that DNA vaccines, which are essentially DNA sequences bound to gold particles and burned in the skin by a helium gun, are effective vaccine delivery systems. Unlike conventional vaccines, these DNA vaccines do not require an additional adjuvant component. In accordance with a further aspect of the present invention, the immunomodulator composition as defined herein may suitably be used in conjunction with such DNA vaccines to increase or participate in the influence of the immune response.

PHARMACEUTICAL COMPOSITIONS

Pharmaceutical compositions comprising a therapeutically effective amount of an entire cell of a bacterium of the genera Rhodococcus, Gordonia, Nocardia, Dietzia, Tsukamurella and Nocardioides and optionally a pharmaceutically acceptable diluent or excipients (including combinations), are also taught in the present document.

The pharmaceutical composition may comprise two components - a first component comprising an antigen and a second component comprising an adjuvant thereof. The first and second components can be administered sequentially, simultaneously or together, and even by different routes of administration.

The pharmaceutical compositions may be for human or animal use in human and veterinary medicine and will typically comprise any or more than one diluent, carrier,

or pharmaceutically acceptable excipient. Vehicles or diluents acceptable for therapeutic use are well known in the pharmaceutical art and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). The choice of the pharmaceutical carrier, excipient or diluent can be selected in relation to the proposed route of administration and conventional pharmaceutical practice.

The pharmaceutical compositions may comprise as -or in addition to-vehicle, excipient or diluent any binder (s), lubricant (s), suspending agent (s), coating agent (s), suitable solubilizing agent (s) ) suitable (s).

Preservatives, stabilizers, dyes and even flavorings can be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents can also be used.

There may be different composition / formulation requirements depending on the different administration systems. By way of example, the pharmaceutical composition of the present invention can be formulated to be administered using a mini pump or by a mucous route, for example, in the form of a nasal spray or spray for inhalation or solution that can be ingested, or potentially in which the compositions formulate by an injectable form, for administration, for example, by an intravenous, intramuscular, intradermal or subcutaneous route. Alternatively, the formulation can be designed to be administered by both routes.

Preferably in the present invention the formulation is in injectable form. More preferably the formulation is injected intradermally.

Preferably in the present invention the formulation is an orally acceptable composition.

When the agent is to be administered orally through the gastrointestinal mucosa, it must be able to remain stable during transit through the gastrointestinal tract; for example, it must be resistant to proteolytic degradation, stable at acidic pH and resistant to the detergent effects of bile.

When appropriate, pharmaceutical compositions may be administered by inhalation, in the form of a suppository or pessary, topically in the form of a lotion, solution, cream, ointment or fine powder, by use of a skin patch, by orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavoring and coloring agents, or may be inject parenterally, for example intravenously, intramuscularly, intradermally or subcutaneously. For parenteral administration, the compositions may be better used in the form of a sterile aqueous solution that may contain other substances, for example, sufficient salts or monosaccharides to make the solution isotonic with blood. For oral or sublingual administration the compositions can be administered in the form of tablets or tablets that can be formulated in a conventional manner.

PHARMACEUTICAL COMBINATIONS

The agent of the present invention can be administered with one or more different pharmaceutically active substances. By way of example, the present invention covers simultaneous or sequential treatments with an immunomodulator composition and / or pharmaceutical composition according to the present invention, and one or more steroids, analgesics, antivirals, interleukins such as IL-2, or pharmaceutically active substance (s)

(s) different.

It will be understood that these regimens include the administration of the substances sequentially, simultaneously or jointly. IMMUNOPOTENCERS

The term "immunopotentiators" as used herein means a

or more bacteria either isolate or in culture that when administered to a subject benefits the health of that subject. Preferably, this benefit is achieved by modifying the subject's cellular immune response.

In accordance with the present invention, immune potencies can be used, for example, for the treatment or prevention of an autoimmune disease or autoimmune disorder.

Immunopotentiators can be administered by consumption in specifically designed foods or animal feed. Immunopotentiators can also be administered by other routes - such as direct injection. Preferably, the bacteria are destroyed so as to avoid the difficulties of maintaining live products.

IDENTIFICATION OF A BACTERIA THAT MODULES AN IMMUNE CELL PHONE RESPONSE

A method for identifying one or more whole bacteria cells of the Rhodococcus, Gordonia, Nocardia, Dietzia, Tsukamurella and Nocardioides genera that modulate (for example, modify) a cellular immune response comprising the steps of: (a) contacting a first test animal with an immune stimulator; (b) contacting a second test animal with an immunostimulator mixed with a bacterium; (c) measure the cellular immune response in each of the test animals; and (d) comparing the cellular immune response in each of the test animals, in which, a minor cellular immune response of the immunostimulator mixed with a bacterium compared to the immunostimulator is only indicative of a modification of the cellular immune response by the bacteria, is described herein.

In addition, a procedure for determining the Th1 / Th2 response of a species of bacteria selected from the Rhodococcus, Gordonia, Nocardia, Dietzia, Tsukamurella and Nocardioides genera is taught, said method comprising the use of the tuberculin skin test. In mice, the tuberculin skin test is preferably carried out on the paw pad. In a predominant Th1 reaction the immune response of the paw pad is maximal at 24 hours and decreased at 48 hours. However, since the reactivity of Th2 increases after 48 hours the positive immune response of the paw pad increases and may even exceed the immune response of the paw pad at 24 hours.

The BCG vaccination effect is well documented using this tuberculin skin test. Thus, the test assay can be used to determine whether or not the introduction of an immunomodulator composition according to the present invention.

modulates the cellular immune response of BCG.

As used herein, the term "test animal" refers to any animal that induces a cellular immune response to the immunostimulator. Preferably, the test animal (s) is (are) a mammal. More preferably, the test animal (s) is (are) a rat, hamster, rabbit, guinea pig or mouse. More preferably, the test animal (s) is a mouse.

Preferably, the bacterium modifies the response of helper T cells. Suitably, the bacterium can modify the auxiliary T cell response by decreasing the Th1 and Th2 response. Suitably, the bacterium can modify the auxiliary T cell response by increasing the Th1 response and decreasing the Th2 response. Suitably, the bacterium can modify the auxiliary T cell response by increasing the Th1 response without affecting the Th2 response.

Preferably, the immunostimulator will have a known Th1 and Th2 response. For example, with the BCG immunostimulator the reaction is usually greater than 24 h when it is an indicator of the Th1 response; the reaction at 48 h is usually lower and includes a contribution of Th2. It is known that BCG predominantly stimulates a Th1 response. By using such immunostimulators it may be possible to determine the Th1 / Th2 response of a test bacterium and, thus, it may be possible to identify one or more bacteria that have a desired Th1 / Th2 response to treat and / or prevent a particular disease and / or disorder.

Preferably, the cellular immune response is measured using the tuberculin skin test. Vaccination with an immunostimulator - such as BCG - induces a test response in the skin with tuberculin (a soluble preparation of Tuberculosis bacilli), when tested later. The local reaction is measured at different intervals, for example, 24 hours, 48 hours and 72 hours after the tuberculin injection. In summary, an immunostimulator (for example, BCG) is used to induce a positive immune response to tuberculin. In the test animal, the tuberculin skin test is preferably carried out on the paw pad. In a predominant Th1 reaction the positive immune response of the paw pad is usually maximal at 24 hours and decreases at 48 hours. However, as the reactivity of Th2 then increases the positive immune response of the paw pad at 48 hours and may even exceed the immune response of the paw pad at 24 hours. Thus, the assay can be used to determine whether or not the introduction of an immunomodulator composition according to the present invention modulates the cellular immune response.

Preferably, the immunostimulator is BCG.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a graph that demonstrates the results of average weights / female litter gain tests and controls during 12 weeks of weaning.

Figure 2 is a graph showing the test results of mean weight gain / litter males and controls during 12 weeks of weaning.

Figure 3 is a graph showing that Gordonia bronchialis enhances the early effect of TH1.

Figure 4 is a graph showing that Tsukamurella inchonensis enhances the early response of TH1 and suppresses the late response of TH2.

Figure 5 is a graph demonstrating that Rhodococcus coprophilus suppresses both TH1 and TH2 responses.

Figure 6 is a graph showing the response to tuberculin for BCG after 1 month compared to unvaccinated controls.

Figure 7 is a graph demonstrating the immune modulation of the effect of BCG by three species of bacteria. Tuberculin responses are measured at 24 and 48 hours. Representative species are (Na) Nocardia asteroides; (Gb) Gordonia bronchialis; and (Tp) Tsukamurella inchonensis.

Figure 8 is a graph demonstrating the immune modulation of BCG / BCG + by two species of bacteria. Tuberculin responses are measured at 24, 48 and 72 hours. Representative species are (Rrh) Phodococcus rhodocrous and (Dm) Dietzia maris.

Figure 9 is a graph demonstrating the immune modulation of the effect of BCG by selected species within the Rhodococcus genus. Tuberculin responses are measured at 24, 48 and 72 hours. (Rrh) Rhodococcus rhodocrous; (Rru) Rhodococcus ruber; (Rrhod) Rhodococcus rhodnii; (Rcop) Rhodococcus coprophilus; (Clothing) Rhodococcus opacus; (Reryth) Rhodococcus erythopolis.

Figure 10 is a graph demonstrating the optimal dose for Rhodococcus ruber using BCG modified with log dilutions of Rhodococcus ruberfrom 108 to 104 compared to BCG alone.

Figure 11 shows a graph of a rat angioplasty study, showing the average weight gain in rat after treatment with Gordonia bronchialis, Rhodococcus corprophilus, or Tsukamurella inchonensis, compared to an untreated control and a control treated with Mycobacteria vaccae .

Figure 12 shows the results for intimate / medium thickening relationships

in rats after treatment with Gordonia bronchialis, Rhodococcus corprophilus, or Tsukaniurella inchonensis, compared with an untreated control and a control treated with Mycobacteria vaccae. GP1 = Control; GP2 = G.bronchialis; GP3 = R. coprophilus; GP4 = T. inchonensis; GP5 = M. vaccae).

The invention will now be described by way of examples, which means that they serve to assist those skilled in the art in carrying out the invention and are not intended in any way to limit the invention in any way.

EXAMPLES

PROCEDURES Tuberculin skin test

The tuberculin skin test is an appropriate test model for determining the effect of an immunomodulator composition, that is, bacterial compositions / suspensions comprising dead whole bacterial cells according to the present invention, in a cellular immune response.

BCG vaccination induces a positive immune response to tuberculin. In mice, the tuberculin skin test is preferably carried out on the paw pad. In a predominant Th1 reaction the immune response of the paw pad is maximum at 24 hours and decreases at 48 hours. However, as the reactivity of Th2 then increases the positive immune response of the paw pad at 48 hours increases and even exceeds the immune response of the paw pad at 24 hours.

The BCG vaccination effect is well documented using a tuberculin skin test. Thus, the test assay can be used to determine whether or not the introduction of an immunomodulator composition according to the present invention modulates the cellular immune response of BCG.

Preparation of a bacterial suspension

Bacterial species of the Rhodococcus, Gordonia, Nocardia, Dietzia, Tsukamurella and Nocardioides genera can be developed in a medium without antigen, such as Sauton medium, in a fermenter for 2-28 days. Alternatively, the bacterial species of interest can be grown on a solid slope. Alternative procedures would be readily available to those skilled in the art.

The resulting bacterial mass can be collected and either used directly and either used directly or after washing to prepare a buffer suspension. The bacterial cell suspension is prepared to contain between 100 nanograms and 10 milligrams of bacilli per dose. Bacterial cells are resuspended in water or saline. Preferably, the saline solution is buffered to pH 8.0 with borate. Preferably the bacilli are inactivated (dead), suitably by heating in an autoclave for 15 minutes at 121 ° C. The resulting bacterial suspension comprises whole cells.

EXAMPLE 1: Modulation of a cellular immune response by Rhodococcus ruber (R.r.).

Group 1: Young adult non-blood female mice were left unvaccinated as a control group.

Group 2: Young adult non-blood female mice were vaccinated on day 0 in the neck of the neck with BCG (105 bacilli) (Evans).

Group 3 :: young adult non-blood female mice were vaccinated on day 0 in the neck of the neck with BCG (105 bacilli) to which Mycobacteria vaccae had been inactivated by heat (M. v.) (107 bacilli).

All mice in Groups 1 -3 were tested on the paw pad to determine the immune response with tuberculin on days 10 and 30. Each mouse was then injected with heat-inactivated heat-inactivated Mycobacteria vaccae (107 bacilli). day 40. On day 50 the tuberculin test was repeated on the paw pad.

Group 4: Young adult non-consanguineal female mice were left unvaccinated as an additional control group. Group 5: Young adult non-consanguineal female mice were vaccinated on day 0 in the neck of the neck with BCG (Evans) (105 bacilli).

Group 6: Young adult non-consanguineal female mice were vaccinated on day 0 in the neck of the neck with BCG (105 bacilli) to which Mycobacteria vaccae (107 bacilli) had been inactivated by heat.

Group 7: Young adult non-consanguineal female mice were vaccinated on day 0 in the neck of the neck with BCG (105 bacilli) to which Rhodococcus ruber (107 bacilli) had been heat-inactivated.

Group 8: Young adult non-consanguineal female mice were vaccinated on day 0 in the neck of the neck with BCG (105 bacilli) to which Rhodoccocus ruber (106 bacilli) had been heat-inactivated.

All mice in groups 4-7 were tested on the paw pad to determine the immune response with tuberculin on day 30 and 30. Then on day 40 each group was divided so that each half of the group received no treatment and second half

He received an injection of Rhodococcus ruber (107 bacilli). On day 50 the test was repeated with tuberculin on the paw pad. The results of Example 1 are shown in Table 1.

TABLE 1: Modulation of a cellular immune response by Rhodococcus ruber (R.r.)

24-hour tuberculin response

48 hours 24 -48 hour difference

Day

1-control group

10

6.9 ± 4.5 5.3 ± 3.7 1.6

30

5.9 ± 4.7 5.2 ± 4.5 0.7

50 M.v.

13.1 ± 5.6 14.0 ± 5.2 + 0.9

p <0.01

p <0.001

Group 2 -BCG

10

16.8 ± 11.1 16.8 ± 5.5 0

30

31.1 ± 17.8 16.9 ± 9.1 14.2 <0.05

50 M.v.

33.9 ± 12.3 19.2 ± 11.3 14.7 <0.02

Group 3-BCG + M.v.

10

14.6 ± 8.3 9.9 ± 2.7 4.7

30

23.8 ± 16.1 21.7 ± 11.5 2.1 n, s

50 M.v.

27.4 ± 10.3 15.1 ± 5.8 12.3 <0.005

Group 4 -control

30

2.8 ± 3.7 2.5 ± 2.3 0.3 n, s,

fifty

6.4 ± 9.4 3.6 ± 7.5 2.8 n, s,

50 R.r.

4.2 ± 5.4 1.0 ± 1.7 3.2 n, s,

Group 5-BCG

30

28.8 ± 15.4 19.1 ± 10.9 9.7 «0.1)

fifty

33.8 ± 21.6 12.0 ± 7.9 21.8 «0.1)

50 R.r.

40.0 ± 22.4 12.2 ± 9.2 27.8 <0.05

Group 6-BCG + M.v.

30

19.4 ± 20.5 17.7 ± 11.8 1.7 n, s,

fifty

56.8 ± 53.8 29.4 ± 31.1 27.4 n, s,

50 R.r.

50.0 ± 38.4 9.0 ± 10.4 41.0 <0.05

Group 7-BCG + R.r.

30

20.6 ± 10.9 17.8 ± 10.3 2.8 n, s,

fifty

24.8 ± 22.5 14.6 ± 13.2 10.2 n, s,

50 R.r.

28.0 ± 13.2 11.0 ± 4.7 17.0 <0.05

Group 8 -BCG + group R.r./10

30

19.5 ± 13.9 20.9 ± 14.9 +1.4

50 R.r.

37.2 ± 25.2 20.2 ± 5.3 17.0 n, s,

fifty

26.8 ± 14.8 12.8 ± 8.0 14.0 (<0.1)

M.v. = Mycobacteria vaccae

In Control Group 1, treatment with Mycobacteria vaccae induced a significant increase in tuberculin response after both 24 hours (p <0.01) and 48 hours (p <0.001). However, in Control Group 4, treatment with Rhodococcus ruberdid

5 does not induce a significant change in the immune response to tuberculin after both 24 hours and 48 hours. In both moments the results of M.v. were significantly greater than the results of R.r. (p <0.02).

In the BCG groups (Group 2 and Group 5), the drop in tuberculin response between 24 hours and 48 hours was greater than (p = 0.06) in mice receiving treatment with 10 Rhodococcus ruber (the average being 28 , 2 ± 15.7) than in mice receiving treatment

with Mycobacteria vaccae (the average being between 14.9 ± 9.6).

In the BCG + Mycobacteria vaccae group (Group 3 and Group 6), the drop in tuberculin response between 24 hours and 48 hours was again between (p <0.05) in the group receiving Rhodococcus ruber treatment ( the average being 41.0 ± 41.0) than in the

15 mice receiving treatment with Mycobacteria vaccae (the average being 12.7 ± 7.0). These data suggest that there is downregulation of the Th2 response after treatment with Rhodococcus ruber, which is not observed after treatment with Mycobacteria vaccae. In the groups of BCG + R.r. (Groups 7 and 8) the effect of the addition of R.r. 107 (Group

20 7) or 106 (Group 8) to BCG was very similar and after the second injection with R.r. there was a substantial reduction in response between 24 and 48 hours (15.5 ± 10.0). EXAMPLE 2: Modulation of a cellular immune response using Nocardia asteroides (N.a.), Gordonia bronchialis (G.b.) or Tsukamurella inchonensis (T.p.).

25 This experiment was designed to compare the test effects of the paw pad with Tuberculin 28 days after vaccination of groups of 6 mice with BCG

alone or with the addition of 107 M. V., R.r., Nocardia asteroides (N.a.), Gordonia bronchialis (G.b.)

or Tsukamurella inchonensis (T.p.). To know the effects that were produced on the immune response to each of these added organisms, the groups of animals were tested on the other pad of the leg 28 days after they were tested with

5 tuberculin with skin test reagents prepared from the body included in its vaccination (Vaccin, Rubin, Asterin, Bronchialin and Inchonensin).

The results are detailed in Table 2. TABLE 2: Modulation of a cellular immune response

10

24-hour tuberculin response

48 hours 24 -48 hour difference

Day

1) Control group

28

3.8 ± 4.8 3.7 ± 3.6 0.1

2) BCG Group

28

38.0 ± 20.2 28.2 ± 17.0 9.8

Tuberculin 56

65.0 ± 31.2 45.8 ± 23.6 19.2

3) BCG + M.v.

28

20.3 ± 10.0 14.2 ± 5.7 6.1

Vaccin 56

18.7 ± 12.1 8.0 ± 6.5 10.7

4) BCG + R.r. Group

28

31.3 ± 16.0 24.2 ± 10.3 7.1

Rubin 56

19.5 ± 7.1 12.3 ± 10.4 7.2

5) BCG + N.a.

28

24.2 ± 20.8 20.2 ± 17.6 4.0

Asterin 56

7.3 ± 8.3 5.3 ± 5.0 2.0

6) BCG + G.b.

28

15.8 ± 14.4 15.7 ± 13.2 0.1

Bronchialin 56

11.3 ± 3.4 6.2 ± 4.3 5.1

7) BCG + T.p.

28

19.5 ± 7.4 15.8 ± 5.7 3.7

Inchonensin 56

9.8 ± 5.0 2.3 ± 2.7 7.5

All bacterial suspensions decreased the response to Tuberculin on day 28 measured at both 24 and 48 hours, compared to that of the next BCG alone (p = 0.05; p = 0.2).

With the exception of Tuberculina and Vaccin, this was the first time that any of

5 these skin test reagents had been used. The differences in sensitivity to the new reagents at 24 hours were probably due to the fact that they had not equilibrated except for protein estimation. However, all showed a drop in response between 24 and 48 hours on day 50, suggesting an immunoregulatory activity.

10 EXAMPLE 3: Local skin reactions to intradermal injections in adult guinea pigs The left side of 3 animals was shaved to provide 0.1 ml intradermal injections containing 108 M vaccae at the anterior end and 0.1 ml containing 109 M vaccinates 5 cm at the end of the animal's tail.

15 Other 3 animals were shaved on the right side and intradermal injections of 0.1 ml containing 108 R. ruber at the front end and 0.1 ml containing 109 R. ruber 5 cm were provided at the end of the tail of the animal.

image 1

In 3 guinea pigs, the local reactions to intradermal injections (the injection route to be used typically in veterinary and medical practice) of 109 R. ruber (a typical dose for human and animal use) were similar to reactions at the same dose of M vaccae in another 3 guinea pigs. At 48 hours after injection, the reactions to R. ruber were more small (p <0.05) than to M. vaccae. There were no differences at 7 days or 14 days. Thus, R. ruber can be even more pharmaceutically acceptable than M vaccae. They do not exist

local reactions to any preparation at the dose of 108.

EXAMPLE 4: Toxicity after subcutaneous injection.

No evidence of toxicity was observed at subcutaneous doses in 17 rats receiving 3 injections of R. ruber when they were 1 day, 14 days and 28 days old.

There were no differences at 7 days or 14 days. Many mice had received injections of various species of bacteria of the present invention, without any evidence of toxicity.

EXAMPLE 5: Determination of bacteria that potentiate Th1 responses only

Adjuvant arthritis is severe experimental arthritis induced in animals by mixtures of oily and mycobacterial extracts (a preparation containing Mycobacteria butyricum is particularly effective). It is believed to require a strong Th1 adjuvant to induce this response. Arthritis is regulated by mechanisms that regulate Th2 down (arthritis requires both Th1 and Th2 mechanisms).

Experiment 1: To determine if R. rubercan induces Adjuvant Arthritis.

Three groups of rats aged 60 -90 days received on the back leg pad:

one.

A suspension in oil of M. butyricum 1 mg / 0.1 ml (n = 8)

2.

A suspension in R. ruber oil 1 mg (n = 8)

3.

An emulsion in oil and saline (n = 8)

Animals of the first group developed arthritis involving the four members, while in rats it provided the suspension in R.ruber oil, only the injected leg was affected.

Experiment 2: Determine if R. ruber can induce a Th2 regulatory response in rats after an exposure of M. butyricum in oil:

To test this, 15 rats were given subcutaneous injections, at different sites, of 107 R.ruber in buffered saline on days -42, -28 and -14 before exposure with M. butyricum in oil emulsion. A control group of 10 rats was previously treated with physiological saline solution following the same program and was subsequently exposed.

The macroscopic evaluation of Adjuvant Arthritis was performed by visual scoring of the individual joints (J Rheumatol 1992; 19: 513-5). Each member was scored 0 -4, with the maximum reaching 16 points / rat.

-42 -28 -14 Day 0 Rro Rro Rro M. butyricum Saline solution Saline solution Saline solution

Both groups did not show differences in the time of appearance of arthritic lesions (usually between 9-11 days after induction), or in the severity of arthritic manifestations. The animals were sacrificed on the 18th for ethical reasons due to the progression of

disease.

conclusion

These experiments clearly show that R. ruber has Th1 adjuvant activity but no "Th2" regulatory activity. Joint damage in Adjuvant Arthritis is a combined action of Th1 and Th2.

EXAMPLE 6: Investigation of the effects of Rhodococcus coprophilus NCIMB 11211, Gordonia bronchialis NC10667 and Tsukamurella inchonensis NC13040 on toxicity, in particular growth rate and subsequent BCG vaccination in

newborn mice

An alternative toxicity test is that subcutaneous injections of Gordonia bronchialis, Rhodococcus coprophilus or Tsukamurella inchonensis are provided on the day of birth 20, and also in a separate site, on day 21. These animals, and controls

injected with saline solution were weighed at regular intervals for 3 months.

Procedures 9 pregnant Balb C mice with terminal phase are used.

25 Dead bacteria suspension preparation: The 10-day culture suspensions prepared in Sauton defoamer broth were centrifuged and resuspended as 10 mg / ml in a borate buffered saline solution, autoclaved and stored at 4 ° C. Dilute 10 mg / ml, 1010 / ml 1/2 in borate to provide 5x109 and then dilute 1/10 in borate buffer to provide a final concentration of 107 in 20 µ for

30 use

Day 1

On the day of birth, remove the newborn mice individually and inject the

litter with 107 dead suspension in the neck and return them to the mother.

Inject the control litter with 20 µl of M15 saline buffered with borate in the neck.

Litter 1 = 2 females + 3 males (Rhodococcus coprophilus NCIMB 11211) Litter 2 = 2 females + 3 males (Rhodococcus coprophilus NCIMB 11211) Litter 3 = 5 females + 3 males (Gordonia bronchialis NC1 0667) Litter 4 = 3 females + 4 males (Gordonia bronchialis NC1 0667) Litter 5 = 1 females + 1 males (Borate buffered saline) Litter 6 = 3 females + 1 males (Borate buffered saline) Litter 7 = 5 females + 3 males (Tsukamurella inchonensis NC13040) Litter 8 = 5 females + 4 males (Tsukamurella inchonensis NC13040) Litter 9 = 4 females + 5 males (saline buffered with borate)

Day 21

Wean the mice, sex them and separate the litters into 2 male and female groups. Weigh and vaccinate again. Mark the individuals in the queue by marking with an indelible pen. Weigh and redial the tail for 3 months. Figure 1 demonstrates the results of average weight gain / litter of females and controls test in the 12 weeks of weaning. Figure 2 demonstrates the mean weight gain / litter test results of males and controls in the 12 weeks of weaning.

The weight gain is the same in all groups, and only 2/57 animals died in the 3 months. Both were males, who died just after getting together with other males after weaning. In this way it can be concluded that the weight gain and progress of the animals was not affected by the three species tested.

EXAMPLE 7: The effect on BCG immunization measured by the tuberculin skin test response

Immunomodulator test model was projected, based on the principle that BCG vaccination induces a response to the skin test with Tuberculin (a soluble preparation of Tuberculosis bacilli), when they were tested 4 weeks later. The local reaction is measured 24 hours, 48 hours and 72 hours after Tuberculin injection. In the mouse, the reaction is usually greater at 24 hours when it is an indicator of the Th1 response to the antigens in Tuberculin. The reaction at 48 hours is usually less and includes a contribution of Th2. The reaction at 72 hours is often a little less than at 48 hours and is a Th2 response. This Tuberculin reaction after BCG can be modulated by prior sensitization, so that the Th1 and Th2 components of the reaction will reflect the nature of the sensitization reagent.

Procedures

Sensitized mice according to Example 6 when they were 3 years old were injected with 106 BCG in 100 µl in the nape in the middle of each trial and in the control group.

At 4 months tuberculin assay with reagent T1475 1 mg / ml. Dilute 100 µl in 1.9 ml to provide a final concentration of 50 µgms / ml. Store at 4 ° C. The dose is 2.5 µg in 50 µ liters provided on the back leg pad. Inflammation of the tuberculin response measured using a micrometer at 24, 48 and 72 hours.

Results

Figure 3 shows that Gordonia bronchialis enhances the effect of TH1 at 24 hours.

Figure 4 shows that Tsukamurella inchonensis enhances the TH1 response at 24 hours and suppresses the TH2 response at 72 hours

Figure 5 shows that Rhodococcus coprophilus suppresses both TH1 and TH2 responses.

conclusion

Each of the 3 species induced different effects in the tuberculin assay after exposure to BCG:

Gordonia bronchialis induced an enhanced Th1 response, without changing the Th2 response. Rhodococcus rubber also has this function.

Tsukamurella inchonensis potentiated the Th1 response and regulated the Th2 response down.

Rhodococcus coprophilus regulated down both Th1 and Th2 responses.

Provided as an adjuvant any of these reagents modulate the immune response to a subsequent antigen.

These results also clearly show that the influence of 2 sensitization immunizations with any of the 3 representative species 3 of the invention persists for at least 9 weeks after the second immunization.

EXAMPLE 8: Immunomodulator test model

Immunomodulator test model was projected, based on the principle that BCG vaccination induces a response to the skin test with Tuberculin (a soluble preparation of Tuberculosis bacilli), when they were tested 4 weeks later. The local reaction is measured 24 hours, 48 hours and 72 hours after Tuberculin injection. The reaction is usually greater at 24 hours when it is an indicator of the Th1 response to the antigens in Tuberculin. The reaction at 48 hours is usually less and includes a contribution of Th2. The reaction at 72 is often a little less than at 48 hours and is a Th2 response. This Tuberculin reaction after BCG can be modulated by prior sensitization, so that the Th1 and Th2 components of the reaction will reflect the nature of the sensitization reagent.

The known immunostimulator, BCG, is injected into the nape of 3-week-old young mice and the tuberculin response was measured 1 month later by subcutaneous injection of tuberculin into the pad of the mouse paw. The resulting inflammation is, the "tuberculin response" is then measured at 24, 48 and 72 hours. Inflammation at 24 hours is considered a Th1 mediated response and inflammation at 48 and 72 hours a Th2 or late mediated response. BCG in the healthy mouse predominantly stimulates a Th1 response.

Process

(a) vial of 10 doses of BCG intradermal vaccine (Evans Medical).

Reconstitute with 1 ml of sterile water supplied using a syringe and needle allowing it to dissolve for 5 minutes. It must be 1x107 / ml.

Using a syringe and needle remove the entire vaccine and transfer it to a bijou plastic bottle.

Dilute 1/10 0.15 ml in 1.35 ml of M15 saline buffered with borate to provide 105 in 100 µ liters

The dose is 105 in 100 µl provided in the neck of the neck.

(b) Tuberculin

T1475 1 mg / ml. Dilute 100 µl in 1.9 ml providing a final concentration of 50 µgms / ml. Store at 4 ° C. The dose is 2.5 µg in 50 µl provided intradermally in the pad of the

hind leg.

The tuberculin response is measured at 24, 48 and 72 hours using a micrometer

Figure 6 shows the response to BCG tuberculin after one month 1 compared to unvaccinated controls.

(c) Preparation of test suspensions

The cultures are grown in Sauton broth, collected by centrifugation and resuspended at a concentration of 10 mg / ml in M15 saline buffered with borate and stored at 4 ° C.

10 mg / ml = 109 in 100 µl.

Dilute 1/10 = 108 in 100 µl.

Add 150 µl of 108 to 1.2 ml of M15 borate and add 150 µl of BCG.

The dose is now 105 BCG + 107 test organism in 100 µl injected in

the neck of the neck

Results

Figure 7 is a graph demonstrating the immune modulation of the effect of BCG by three species of bacteria. Tuberculina responses are measured at 24 and 48 hours. Representative species are (Na) Nocardia asteroides; (Gb) Gordonia bronchialis; and (Tp) Tsukamurella inchonensis.

Figure 8 is a graph demonstrating the immune modulation of BCG / BCG + by two species of bacteria. Tuberculina's responses are measured at 24, 48 and 72 hours. Representative species are (Rrh) Rhodococcus rhodocrous and (Dm) Dietzia maris.

Figure 9 is a graph demonstrating the immune modulation of the effect of BCG by selected species within the Rhodococcus genus. Tuberculina's responses are measured at 24, 48 and 72 hours. (Rrh) Rhodococcus rhodocrous; (Rru) Rhodococcus ruber; (Rrhod) Rhodococcus rhodnii; (Reop) Rhodococcus coprophilus; (Clothing) Rhodococcus opacus; (Reryth) Rhodococcus erythopolis.

Figure 10 is a graph demonstrating the optimal dose for Rhodococcus ruber using BCG modified with Rhodococcus ruberfrom log solutions 108 to 104 compared to BCG alone. conclusion

This screening test demonstrates that mixing the test suspensions with BCG and comparing with BCG alone, 28 days after the tuberculin test, as a measure of response to BCG, has been regulated or modified. In this simple model, immunoregulation is shown as a depression in the size of the response at both 24 and 48 hours. This can also be investigated in longer-term experiments in which sensitization with the test suspension is carried out a few weeks before exposure with BCG and subsequent Tuberculin assay.

EXAMPLE 9: To show that whole cell suspensions of bacteria are more effective in modulating immunity than cell extracts, comparative preparations are prepared and tested in a skin test system for exposure to BCG / Tuberculin .

100 mg of Tsukamurella inchonensis are suspended in buffered saline solution (pH 8.0) in a pre-weighed centrifuge tube at a concentration of 10 mg wet weight of organisms / ml. The complete 10 ml treated in an ultrasonic to break most organisms (70 -80%).

The sonicate is centrifuged at 15,000 rpm for one hour in a tube and the supernatant removed 0.2 µn as the test extract. The tube, with deposit, is weighed again to determine the proportion of whole organisms that have not broken any more cell wall debris etc. This is 64mg. The volume of extract equivalent to 0.01 mg of T. inchonensis is estimated later and this is compared with the equivalent of 0.01 mg (approximately 107) of whole bacilli.

Groups of 10 animals receive injections of the extract or suspensions of whole bacillus cells (equivalent to 0.01 mg / dose) or placebo of buffered saline in the neck of the neck at weaning and 7 days later. Two weeks later the animals were exposed with BCG. 28 days later. The Tuberculin test is performed with readings taken at 24, 48 and 72 hours.

Results:

The tuberculin responses in mice after BCG Vaccination provided after sensitization with nothing, borate buffer, whole T.inchonensis cells or soluble antigens (sonicated from the filtrate).

The response to Tuberculin after BCG provided to non-sensitized animals:

Nº 24 hours 48 hours 72 hours 6

9 ± 4.2 7.7 ± 3.39 3.67 ± 3.5

The response to Tuberculin after BCG provided to animals sensitized with borate buffer alone:

6 9 ± 5.7 6.3 ± 7.1 3.17 ± 2.93

5 Sensitization with borate buffer had no effect on the BCG Tuberculin assay. The response to Tuberculin after BCG provided to heat-inactivated T. inchonensis sensitized animals:

6 7.3 ± 1.03 6.17 ± 3.87 1.5 ± 2.07

10

Sensitization with whole T. inchonensis decreases Tuberculin responses, notably at 72 hours (Th2 sensitivity). The response to Tuberculin after BCG provided to animals sensitized with soluble preparation of T. inchonensis (sonic filtrate):

fifteen

6 6.8 ± 6.8 11.0 ± 8.0 4.6 ± 6.8

Sensitization with sonic filtration of T. inchonensis increases responses at 48 hours and 72 hours (Th2 sensitivity). This is probably due to an increase in the production of inflammatory antibodies to share antigens between BCG and T.

20 inchonensis The results show that sonic sensitization results in a response that is different from sensitization with whole dead organisms. Preliminary research suggests that whole bacteria cells are more effective in modulating immunity than cell extracts.

25 EXAMPLE 10: Study of angioplasty of rat model of vascular disease

The model is based on an induced reduction of thickening of the layers of the

intima of the common carotid artery of rats observed after balloon angioplasty. The experimental groups consisted of 15 male rats each: Group 1 were rats injected with 0.1 ml of buffered saline solution with

30 borate at pH 8 subcutaneously (CONTROL) Group 2 were rats injected with 0.1 ml of borate buffered saline at pH 8 subcutaneously containing environmental saprophytic bacteria Gordonia bronchialis

heat inactivated

Group 3 were rats injected with 0.1 ml of borate buffered saline solution at pH 8 subcutaneously containing heat-inactivated Rhodococcus coprophilus environmental saprophytic bacteria

Group 4 were rats injected with 0.1 ml of borate buffered saline solution at pH 8 subcutaneously containing heat-inactivated Tsukamurella inchonensis environmental saprophytic bacteria

Group 5 were rats injected with 0.1 ml of borate buffered saline solution at pH 8 subcutaneously containing heat-inactivated Mycobacteria vaccae environmental saprophytic bacteria (positive control).

Experimental procedure time course:

All animals were allowed 7-10 days to accommodate before the start of the experiment on Day 0. Day 0 was weighed at weekly intervals after this as a measure of well-being.

Rats in all groups were fed with a standard rat feed and free access to water was allowed at will.

On day 0 the rats were injected subcutaneously with control or active agent as described above (the organism dose was 50 micrograms in 0.1 ml, that is, 500 micrograms / ml)

On day 21 the rats were injected with a second dose of control / active agent subcutaneously (the organism dose was 100 micrograms in 0.1 ml, i.e. 1 mg / ml)

On day 49, 0.75 ml of blood was collected from the tail vein of each rat and stored properly to allow the cytokine RNA to be measured later.

On day 56 all the rats underwent balloon trauma in the left common carotid artery under anesthesia.

On day 70 all the rats were weighed and sacrificed by a Program 1 procedure and the carotid arteries, spleen, left kidney, part of the left lung, part of the middle lobe of liver, heart and thoracic aorta were collected for immunological analysis. Blood was also collected for further analysis.

Transverse sections of each left carotid were cut and stained with hematoxylin and eosin. The muscular and intimal layers were measured separately microscopically and expressed as areas of the cross section of the vessel. Additional sections for immuno-histochemistry will be used to determine the types of immune cells that are present in the cytokines that are being released.

Blood samples were collected on days 49 and 70 were stored in a manner suitable for subsequent analysis of cytokine RNA, chemokines and other substances related to vascular disease.

Results

The results of rat body weights are shown in the Table below and also in Figure 11. Table: Rat weights in g between day 0 and day70 of example 10

Group

Day

0

7 14 2 28 35

one

352 368 380 392 400 417

2

359 377 392 409 424 437

3

350 366 381 395 406 417

4

360 376 388 402 411 422

5

353 370 385 395 408 417

Day

Angioplasty

42

49 56 63 70

424

434 437 413 428

443

456 460 447 460

424

433 439 422 439

428

439 443  428 446

427

433 438 424 443

Day

Angioplasty

1v2

1v2

P <0.03

P <0.01

Animals sensitized with injections of Gordonia bronchialis (2) show a significant weight gain over the placebo group (1) in two moments

The results of the intimate and average thickness measurements are shown in the

Table below and the results for Intimate / average ratios ratios are shown in the

figure 12.

Table: The mean values for the intimate and average in different groups of Example 10.

Intimate Medium

Placebo 118233 ± 48506 125434 ± 35064 n = 31 *

G bronchialis 65167 ± 25068 131833 ± 39016 n = 41

R. coprophilus 89506 ± 35109 149469 ± 7913 n = 34 T.inehonensis 66153 ± 37546 147426 ± 17494 n = 28 M.vaccae 50924 ± 27363 139523 ± 20206 n = 38

* number of sections studied

Differences between Placebo values

G. bronchialis

p <0.001 n. s.

R. coprophilus.

p <0.01 p <0.001

T. inchonensis

p <0.001 p <0.005

M. vaccae

P <0.001 p <0.05

10

Highly significant differences are observed between receptors of active agents and placebo. The reduced results for intimacy reflect a reduction in myointimal hyperplasia after angioplasty. This occurs with the four bacterial preparations, the most

15 notable with G. bronchialis, T. inchonensis and M. vaccae. This effect could make a great contribution to the success of angioplasties.

The average thickness loss in the placebo group is maintained in the active treatment groups, most notably after R. coprophilus and T. inchinensis. The combination of reduced intima thickness and maintained average thickness, observed better after treatment with T. inchonensis, can prove important progress in the

potential reduction of chronic graft rejection.

EXAMPLE 11: Myocarditis in rats infected with Trypanosomiasis

Animal preparation

5

10

fifteen

twenty

25

30

40

i) On day one "1" old male rats were injected into the neck of the neck with 107 Gordonia bronchialis, Rhodococcus ruber, Rhodococcus coprophilus, Tsukamurella inchonensis or Mycobacteria vaccae in a volume of 0.1 ml by subcutaneous injection. (This was carried out in two experimental rounds, as shown in the table below).

ii) After 14 days, male rats "1" were given a second subcutaneous injection with 107 of the same organisms in 0.1 ml on the left side. iii) The animals were exposed with Trypanosoma cruzi alive on day 21 subcutaneously with 106 trypomastigotes of the Tulahuen strain of T. cruzi. Infectious blood trypomastigotes were maintained by serial passage in CBi mice. iv) Forms of the circulatory torrent of T. cruzi will be assessed under conventional conditions, by direct microscopic observation of 5 µI of venous blood from the heparinized tail, on days 7 and 14 days after infection (pi). Data are expressed as number of parasites / 50 fields. v) 7 days later an additional subcutaneous injection of 107 of the same organism was administered in a volume of 0.1 ml on the right side.

Control animals only received provocation with T. cruzi. Another group of animals were left unprovoked for comparison purposes.

PARASITEMIES

Day 7

Day 14

GROUP

n Mean ± SD Medium (category) Mean ± SD Medium (category)

T. cruzi control

14 2.35 ± 1.44 2 (1 -5) 1.07 ± 1.07 1 (0 -3)

M. vaccae

17 1.17 ± 1.01 1 (0 -3) 1.29 ± 2.1 0 (0 -7)

R. ruber

17 0.65 ± 0.78 0 (1 -2) 0.69 ± 1.01 0 (0 -3)

P <0.01 for M. vaccae and P = 0.001 for R. ruber compared to the T. cruzi control

Animal preparation

On day one old male "1" rats were injected into the neck of the neck with 107 Gordonia bronchialis, Rhodococcus coprophilus, or Tsukamurella inchonensis in a volume of 0.1 ml by subcutaneous injection. (This was carried out in two experimental rounds, as shown in the table below).

After 14 and 28 days, male rats "1" were given a second and third subcutaneous injection with 107 of the same organisms in 0.1 ml on the left side.

The animals were provoked with Trypanosoma cruzi alive on day 21 subcutaneously with 106 trypomastigotes of the Tulahuen strain of T. cruzi. The blood trypomastigotes

Infectious diseases were maintained by serial passage in Cbi mice. Control animals only received exposure with T. cruzi. Another group of animals was left for comparison purposes.

5

Day 7

Day 14

Control Group of T. cruzi G. bronchialis R. coprophilus T. inchonensis

n 6 4 5 6 Mean ± SD 3.83 ± 4.36 3.25 ± 3.3 1.6 ± 0.89 3.83 ± 5.64 Mean (category) 4.5 (1 -22) 2 (0-8) 3 (1-8) 5 (0 -20) Mean ± SD 3.5 ± 2.89 0.33 ± 0.58 1.0 ± 1.73 2.0 ± 1.83 Mean (category) 2 (0 -6) 0.5 (0 -5) 0 (0 -3) 2 (0 -18)

P = 0.032 on day 7 and P = 0.05 on day 14 for R. coprophilus compared to the control of T. cruzi (analysis of Kruskall-Wallis variance).

Serum IgG antibodies specific for T. cruzi 28 days after infection with

T. cruzi

Optical density values of the ELlSA test

T. cruzi control

0.3

G. bronchialis

1.4

R. coprophilus

0.5

T. inchonensis

0.9

CD4 + cells in the myocardium 7, 14 and 21 days after provocation with T. cruzi.

Day 7 p.i.

Day 14 p.i. Day 21 p.i

T. cruzi control

26.4 ± 4.5 25.6 ± 8.4 44.6 ± 10.5

G. bronchialis

48.6 ± 7.5 63.4 ± 8.2 74.3 ± 10.8

R. coprophilus

53.2 ± 9.9 50.0 ± 8.1 32.9 ± 8.6

T. inchonensis

40.0 ± 8.5 38.6 ± 8.2 44.5 ± 9.2

The heart muscle of additional test subjects will be analyzed 3 months after infection with T. cruzi for a determination of chronic myocarditis. Preliminary research has shown that Mycobacteria vaccae has a beneficial effect in reducing chronic myocarditis and it is expected from previous data that some test organisms have beneficial effects in preventing chronic myocarditis.

EXAMPLE 12: Use of T. inchonensis to reduce myointimal hyperplasia (MIH) after coronary angioplasty

Atherosclerotic vascular disease is the most common cause of death and disability in the world. The development of atherosclerosis is a complex process, with lipid deposition that leads to narrowing of arteries and endothelial dysfunction. The evidence is the development that inflammatory responses at vascular lesion sites play a key role in the disposition to atherosclerosis (Schett G, et al. J Clin. Invest. 1995, 96: 2569-2477). Atherosclerotic plaque rupture is the most common cause of sudden arterial occlusion that leads to acute ischemic syndromes such as myocardial infarction, stroke and peripheral arterial occlusion. After an acute ischemic episode, a number of inflammatory markers are elevated including certain cytokines and C-reactive protein (CRP) (George et al Circ. Res. 2000; 86: 1203-1210; Hansson et al Arteriosclerosis 1989; 9: 567 - 578; Yokota et al J Intern Med. 1995; 238; 479-489; Ikeda J. Mol. Cell. Cardiol. June 24, 1992 (6): 579 584; Hojo et al Heart July 2000; 84 (1) 83 -7.

The expression of stress proteins, even at sites of complete intimate stress damage, leads to a local immune response with cytokine release in the middle and intimate according to the time of the immune response that predominates in the individual in time (Chan et al Eur J. Vasc. Enodvasc. Surg. Nov. 1999 18 (5): 381-5; Mukherjee et al Thromb. Haemost 1996; 75: 258-260; Wright Heart Vessels 2000; 15: 18 -22; Sanchez-Margalet et al Clin Chem Lab. Med August 2002; 40 (8); 769-774). Some cytokines lead to intimal thickening and a phenotype change in the smooth muscle cells of the media, together resulting in myointimal hyperplasis (MIH). Local release of other cytokines results in intimate repair with minimal obstruction of blood flow (Hansson et al Proc. Natl Acac. Sci. United States 1991; 88: 10530-10534; D'Elios et al Transplant Proc 1998 ; 30: 2373-7; Feldman et al Circulation February 29, 2000; 101 (8): 908-916; Tashiro Coron Artery Dis March 2001; 12 (2): 107-13; Hojo et al Atherosclerosis May 2001 ; 156 (1): 165-170; Yang et al Circulation March 7, 2000; 101 (9); 1019-26; Anguera et al Am. Heart J. Nov. 2002, 144 (5): 811-817; Takase et al Can J. Cardiol, July 2003; 19 (8): 902-906; Tutar et al Circulation September 30, 2003; 108 (13): 1581-1584 E-pub September 15, 2003; Del Prete et al Blood July 1, 1995; 86 (1): 250-257). The purpose of our proposal is to ensure that the last mechanism is on site.

The link between myointimal hyperplasia (MIH) and inflammation:

The immune mechanisms that lead to the development and maintenance of MIH result in an increase in stress associated with restricted blood flow, the establishment of fatty stretch marks and atherosclerosis plaques. Increase in IgG2 antibody levels to stress proteins expressed on minimally damaged intimal cells results in local induction of the complement cascade and destruction of damaged cells (Schett et al (supra); Chan et al (supra); Crisp et al J. Herat Lung Transplant 1994; 81 -91; Johnson et al. Atherosclerosis 1990; 84: 111-119). On this site, T lymphocytes are attracted to initiate local cytokine production (Hansson 1991 (supra); D'Elios (supra); Mickelson et al J Am Coll Cardiol. 1996 Aug; 28 (2): 345-53) . A fatty streak is established on the damaged intima under which the cytokine activity, beneficial or prejudicial, takes place in a manner that determines atherosclerotic repair or progression. As a non-specific result of local inflammation, interleukin cytokines (IL) -1 beta and tumor necrosis factor alpha (TNF-a) induce IL-6 which in turn potentiates the biosynthesis of proteins in acute phase (for example, Fibrinogen, CRP) by hepatocytes (Ikeda et al (supra); Hojo et al (supra); Spaziani et al Ital Heart J. October 2002: 3 (10): 593-597).

MIH can be considered as an exaggerated healing response to vascular injury after percutaneous coronary intervention (PCI). A cascade of episodes, which eventually leads to stenosis or occlusion of the vessel, can occur, including:

•

loss of the basement membrane

•

Migration of vascular smooth muscle cell (VSMC) cells from the middle to the intimate.

•

Proliferation of VSMC and phenotypic change to a more secretory fibroblast cell type

•

Increase in extracellular matrix production

Clinical restenosis occurs after percutaneous coronary intervention (PCI) balloon angioplasty and affects approximately 30% of such cases in clinical practice. It is the main cause of failure of such procedures and treatment of the vessels / grafts with stenosis and resulting blockages is problematic. The underlying cellular mechanisms that lead to MIH are not well understood although they are associated with a predominance of auxiliary T-cells type 2 (Th2), which produce the cytokines IL-4 and IL-5, in the presence of which TNF-a arrives to damage tissue (Hernandez-Pando et al lmmunology 1994; 82: 591-595). To date, no therapy has been developed that can effectively avoid the process. However, the role of a locally mediated inflammatory response that can later become a more systemic phenomenon is accepted as a key part of the PCI failure. The clinical relevance of the complete project refers to the very numerous percutaneous coronary interventions that are performed annually in the United Kingdom UK (approximately 40,000) and worldwide. The new generation of permanent vascular dilators that elude drug have many lower percentages of restenosis than permanent vascular dilators "of unprotected metal", but are unlikely to avoid restenosis completely and are expensive. A safe, relatively inexpensive, complementary therapy to prevent MIH, such as the immunotherapy proposed in this protocol, could have a significant clinical impact.

Certain bacterial species possess the ability, when dead, to regulate the immune response to antigens, such as heat shock proteins, presented with them. Suspensions of such dead bacteria are very safe to use, and the desired immune modulation can be obtained using various species. Recent studies in our laboratory show that patients with atherosclerosis may have decreased Th1 immunity compared to controls of the same age. In this way inflammatory responses to vessel injury can be modified by altering the response pattern by appropriate immunotherapy.

There has been much interest in the role that Th1 and Th2 cytokines play in various other human diseases such as tuberculosis, asthma, rheumatoid arthritis and a number of autoimmune conditions (D'Elios M, Del PG. Transplant Proc 1998; 30: 2373 - 7; Shirakawa T, et al. Science 1996; 275: 77-79; and Hernández-Pando R, Rook GAW. Immunology 1994; 82: 591-595). Th1 cells produce IFN-y, interleukin-2 (1 L-2) and TNF. Th2 cells produce IL-4, IL-5, IL-6, IL-10 and IL-13 (Lin E, et al. Surgery 2000; 127: 117-126). Both types of T cells can cause cell-mediated inflammation, and both lead to antibody formation, although different subclasses of immunoglobulin are involved, and the amount of antibody that accompanies a Th2 response is usually much greater. When regulation of Th1 responses fails, Th1-mediated autoimmune diseases such as multiple sclerosis can occur (Genain CP, et al. Science 1996;

274: 2054-2057). Unregulated Th2 responses can lead to a variety of pathologies, including allergic reactions, Th-2-mediated autoimmunity and also chronic fibrotic inflammation such as that seen in idiomatic pulmonary fibrosis (IPF) (Wallace W, et al. Clin. Exp. Immunol. 1995; 101: 436-441; and Du Bois RM. New Engl. J. Med. 1999; 341: 1302-1304).

Such TH2 responses to vascular smooth muscle cell (VSMC) and endothelial antigens and play a similar causal role in clinical MIH.

In some situations, such as tuberculosis and rheumatoid arthritis, simultaneous production of Th1 and Th2 cytokines may result in additional tissue damage. At least one mechanism for this is the increased necrotizing activity of TNF-ү tissues associated with Th1 in the presence of Th2 cytokines IL-4 and IL-5. The part played by such combined activities is uncertain in MIH, but mycobacterial diseases such as tuberculosis and Johne cattle disease, in which it occurs, are known to predispose to atherosclerosis (Alibasoglu M, et al. Am J Vet Res. January 1962; 23: 49-57).

Inflammation is now linked to the progression of atherosclerotic vascular disease. Vascular lesion, which occurs naturally by rupturing the spontaneous plaque, or by angioplasty, stimulates inflammatory responses that may adversely affect healing and cause myointimal hyperplasia. A preparation derived from a heat-inactivated bacterium according to the present invention modulates the immune response to vascular injury in patients undergoing percutaneous coronary intervention for symptomatic coronary artery disease. The inflammatory response measured by venous blood levels of antibodies to stress proteins, a range of cytokines, cortisol, dehydro epi-androsterone (DHEA) and C-reactive protein will be modified in the subjects to whom active preparation is provided. . A sample size of 50 patients will be sufficient to detect differences of approximately 20% in the levels of these markers.

Immune modulation may affect the inflammatory response: The mode of action of heat-inactivated T. inchonensis is believed to be a regulator of maturation of T cells in relation to antigens shared between bacterial preparation and those of human tissues . Notably, these include 60 and 70kDa stress proteins of mitochondrial origin known to share some 60% of their amino acid chain length homology with bacterial 65 and 70kDa heat inactivated proteins. The regulatory activity of T. inchonensis is to decrease the mechanisms of Th2 and enhance the mechanisms of Th1, changing the response to intimate expression of stress proteins, and regulating the VSMC phenotype through the local production of cytokines. A predominant Th2 response with high antibody titers to stress proteins leads to the destruction of intimate cells that express these proteins through the complement cascade, with local release of type 2 cytokines (IL-4, IL-5 and IL-13), modifying the VSMC phenotype for rapid replication. With a Th1 response, intimate cells that express stress proteins are thought to be destroyed individually by T cells releasing type 1 cytokines (IL-2, IFN-a, and TNF-a) inducing less phenotypic changes proliferative in VSMC and local tissue repair. The first of these mechanisms would lead to additional damage and restenosis, while the second should lead to intimate repair without stimulation of restenosis.

Such changes in the activity of Th cells accompanies a decrease in plasma cortisol with an increase in plasma DHEA, and an effective regulation of inflammation that occurs in a return to normal values for C-reactive protein etc.

Although not wanting to be linked to any theory, an autoimmune target in the vessel pairs may be the heat shock protein (HSP) 70.

The mechanisms by which immunotherapy modifies the inflammatory response after vascular injury are not fully understood, but it is believed that the combination of specific adjuvant activity of T. inchonensis, together with antigens shared between the bacilli and tissues, result in immunity regulation so as to reduce the damage of immunized tissue. Endothelial injury caused by angioplasty can be increased by the host's immune response to HSP. These HSPs in the closely similar form of stress proteins are produced by stressed cells that have been implicated in the pathogenesis and pathophysiology of various immune disorders including atherosclerosis (Xu et al Arterioscler. Thromb. 1992; 12: 789-799). They are likely to be present in endothelial and smooth muscle cells in the region of an angioplasty. Indeed, the hsp / stress protein acts as a self-antigen, which can then be directed by the immune system in an appropriate manner. This situation can be experimentally induced by immunization with a myobacterial cross-reaction hsp (hsp65) that leads to endothelial damage in rabbits and mice. The effect appears to be dependent on IL-4 secreted by Th2 lymphocytes, and is probably antibody-mediated, as described above.

The relevance of these observations from animal animal studies to man is suggested by the ability of human antibody purified by eluted affinity of hsp65 columns to damage human endothelial cells stressed in vitro (Schett et al (supra)). This finding suggests that the antibody cross-reacts with hsp60, which is the human homologue of hsp65, and may be accessible to the antibody when expressed on the membranes of stressed endothelial cells. It has been suggested that such antibodies that bind to stressed endothelial cells may be a factor in the production of coronary artery disease after heart transplantation (Crisp et al (supra)). Mukherjee et al. They showed no association between preoperative antibody levels with hsp65 and coronary restenosis, but showed that those patients where levels of such antibodies fell after angioplasty were less likely to have restenosis.

(Mukherjee et al (supra)). They did not determine the IgG subclass to which the antibodies to which hsp 65 belonged, and the reduced levels recorded could have been due to change to IgG4 (Th1 associated antibody). In fact, the role of antibodies to hsp can be complex, because patients with vascular disease not only had increased antibody, but also had elevated levels of hsp / stress proteins themselves (Wright et al (supra )). Thus an obvious drop in antibody levels can only reflect an increase in the levels of the protein or antibody that binds to the protein. In addition, hsp / stress proteins have regulatory effects, and bind to arterial smooth muscle cells, which leads to enhanced survival without a requirement for internalization (Johnson et al (supra)).

Pretreatment of patients due to undergoing percutaneous coronary intervention with an immunomodulatory reagent prepared from heat-inactivated Tsukamurella inchonensis will reduce inflammatory processes, reducing the production of local myointimal hyperplasia and subsequent formation of atheroma and artery restenosis. This will be recognizable by changes in the cytokine content in the peripheral blood using the very sensitive "Luminex" procedure, measuring 22 different cytokines. It will also be detectable by changes in the levels of certain stress proteins and antibodies to them, and by changes in serum cortisol, dehydroepiandrosterone (DHEA), and their derivatives.

To study the effect of injections of a heat-inactivated preparation of Tsukamurella inchonensis on (i) inflammatory response, measured by the levels of Interferon-y, TNF-α, IL-6, IL-10, (ii) other regulatory measures by auxiliary T cells and function, (iii) levels of circulating stress proteins and antibodies to them, (iv) protein, and (5) plasma levels of cortisol and dehydro epiandrosterone, observed after elective percutaneous coronary angioplasty (PCI) ; a randomized double blind controlled parallel group study will be used.

Patients requiring PCI for clinical reasons can be chosen to take part. The study size is 50 patients that will be randomly distributed for immune therapy with T inchonensis or an inactive vehicle. Patients will receive injections, 6 weeks and 3 weeks before angioplasty and a third injection 4 weeks after the procedure. The markers of inflammation will be measured before the first injection, just before the procedure, 24 to 48 hours after the procedure and 6-8 weeks after the procedure.

The study will have 2 phases; In the first 10 randomized patients who are going to receive active product or placebo, all the mentioned parameters will be investigated. In the second phase, the remaining patients will be investigated by procedures selected as the most appropriate of the first stage.

The first injection will be given intradermally in the upper arm

or shoulder area 6 weeks before the scheduled angioplasty and a second injection will be given 3 more weeks. The procedure will be done 3-5 weeks after the second injection, and a third and final injection will be given 4 weeks after the procedure. The PCI should take place as routinely as indicated and should be followed up at 6 -8 weeks that should coincide with the appropriate clinical appointment. Blood samples for immunological and inflammatory markers should be taken at the beginning of the experiment (before the first injection of immune or placebo therapy), immediately before the PCI procedure, 24-48 hours after the PCI procedure, and 6 - 8 weeks after the PCI procedure.

Blood sampling protocol: 10-15 ml samples of venous blood will be collected on each of the 4 occasions. These will be for routine hematological and immunological tests, for a battery of serum cytokine and chemokine determinations, for the measurement of circulating stress protein levels and antibodies for them, for the determination of cortisol and DHEA in plasma, for the measurement of inflammatory markers and vascular disease and for RNA storage for analysis of intracellular cytokine production.

Details of immune therapy preparation: The preparations will be M / 15, physiological saline without pyrogen buffered with borate (pH 8) (placebo), or a suspension of heat-inactivated organisms of the aerobic actinomycete species of Tsukamurella inchonensis, suspended at 10 mg / ml in the same borate buffer.

The bacilli developed on a non-antigenic liquid medium, a non-animal product, (approved by Veterinary Medicines Directorate as well as animal viruses). The incubation was in a shaking water bath at 32 ° C. Bacilli were collected when good growth was obtained after 10 days of incubation.

The placebo and bacterial suspension were distributed in sterile 2 ml multidose vials, in 0.5 ± 0.1 ml volumes for individual dosing. The vials were autoclaved at 121 ° C for 15 minutes in a regularly maintained autoclave with efficacy indicators. After cooling, the vials were labeled and stored at 4 ° ± 1 ° C in boxes dedicated to the study.

Randomization: This will be through the production of random sequence generated by computer number, 1 to 10 and 11 to 60 (covering patients who are out or road accidents).

Vial marking can be with the patient study number and A, B, or C to indicate if it will be used for the first, second or third injection (a self-adhesive disposable label will be attached to each vial that can be attached to the patient's notes against the administered data). The placebo and active product vials will be marked or labeled

similarly in order to use in the boxes, following a computer generated randomization scheme. The administration of the interventions will be by intradermal injection into the skin in the alternative deltoid muscles, beginning with the left shoulder. The right vial for

5 the patient will be taken from the refrigerator. Immediately before putting in a syringe, the vial will be vigorously shaken for 20 seconds to suspend the particulate bacilli. Then 0.1 ml will be removed in an insulin syringe "BD Microfine + 1 ml of U-1 00 with a 0.33 mm (29G) x12.7 needle (fused)", and immediately administered, taking care to lift A small area of orange peel. The full dose should be injected

10 of 0.1 ml. After the injection, the remaining vial with its contents will be returned to a special box in the refrigerator for future checking, if necessary.

Table of Procedures

Procedure / episode

Base line Time 0 3 weeks 6 weeks + 1 -2 days 10 weeks 14 weeks

Admissibility check

image2

Consent

Randomization

History and physics

Trial intervention

Check Injection Site

image2 image2 image2 image2

Blood sample

image2

PCI

Adverse episodes check

image2 image2 image2 image2

Final tracking

15 Study interventions will be performed 3 times, 2 before angioplasty and 1 after. The first 2 should be with 21 ± 3 days of separation to angioplasty should follow the second injection in 28 -52 days and the third injection of study treatment should be 28 ± 3 days after angioplasty (i.e. 52-80 days after the second injection). Study results: The main outcome of interest are serum markers that

20 reflect immune modulation and changes in the levels of acute phase reactions such as

inflammation indicators Blood samples will be analyzed for the determination of a diversity of markers but the measurements of the main result of the study will be a comparison of the differences detected between the two treatment groups in the follow-up of the blood samples compared to the samples of the line base, and with the sample taken before PCI.

The following investigations will be carried out on the 10 patients in phase 1 of the study:

one.

Determination by luminex of 22 different cytokines.

2.

Cortisol, DHEA and plasma metabolites.

3.

Serum levels of 60/65 kDa and 70k Da hsp / stress proteins, and antibodies to them.

Four.

C-reactive protein levels.

5.

Compliance activation.

6.

Markers of vascular disease.

7.

Routine hematology etc.

8.

Pax-gen system studies for intracellular cytokines will be performed if the results of the previous tests are inadequate. The investigations to be carried out in the second and subsequent stages of the test are selected from the results obtained from phase 1, as described below.

The results obtained for the first 10 patients will be analyzed by each of the researchers to:

one.

Those that show differences between blood samples 1 and 2 (immune modulation effect).

2.

Those that show differences between blood samples 2 and 3 (effect of operative procedure).

3.

Those that show differences between blood samples 1 and 4 (effects of treatment and procedure).

The results will then be analyzed according to patients' active or placebo treatment. Selected investigations that show differences between these 2 groups will be applied to the second, main phase of the study.

Secondary results: Aliquots of blood samples will be stored for possible subsequent analysis of other vascular inflammatory markers, such as endothelin, von Willibrand factor etc.

Preliminary research suggests that T. inchonensis reduces hyperplasia of

Myointima (MIH) post coronary angioplasty.

Claims (20)

 Claims

one.

Use of an immunomodulator composition or a pharmaceutical composition comprising an entire cell of a bacterium of the genus T. sukamurella in the manufacture of a medicament for the treatment or prevention of an autoimmune disease or autoimmune disorder.

2.

Use according to claim 1 wherein the autoimmune disease or autoimmune disorder involves inflammation of the intima of a blood vessel.

3.

Use according to claim 2 wherein the autoimmune disease or autoimmune disorder further involves inflammation of the muscular layer of a blood vessel or myocardium.

Four.

Use according to any one of the preceding claims wherein the autoimmune disease or autoimmune disorder is one that is preceded or caused by a vascular disorder.

5.

Use according to any one of the preceding claims wherein the autoimmune disease or autoimmune disorder is selected from one or more of the following: arthritis, particularly rheumatoid arthritis, psoriasis, psoriatic arthropathy, scleroderma, thyroiditis, intimal hyperplasia after transplantation, graft rejection and vascular disorders, in which the vascular disorder is a vascular disorder that comprises an autoimmune element.

6.

Use according to claim 5, wherein the vascular disorder is selected from one or more of the following: Raynaud's disease or phenomenon, anterior uveitis, obliterating vascular disorder, atheroma formation, arteritis, myointimal hyperplasia, inflammatory thickening and autoimmune of the intima and / or muscular layer of blood vessels, inflammatory lesions of blood vessels, atherosclerotic heart disease, reperfusion injury, cardiac conduction abnormalities, myocarditis, myocardial infarction.

7.

Use according to claim 5 wherein the graft rejection is chronic graft rejection.

8.

Use according to claim 5 wherein the graft rejection is chronic graft rejection in the absence of immunosuppressant.

9.

Use according to any one of claims 1-8 wherein said bacterium is selected from one or more of the group consisting of Tsukamurella paurometabola and Tsukamurella inchonensis.

10.

Use according to any one of the preceding claims wherein said bacterium is dead.

eleven.

An immunomodulator composition or pharmaceutical composition comprising a

whole cell of a bacterium of the genus Tsukamurella, for use in the treatment or prevention of an autoimmune disease or autoimmune disorder.

12.

An immunomodulator or pharmaceutical composition according to claim 11 wherein the autoimmune disease or autoimmune disorder involves inflammation of the intima of a blood vessel.

13.

An immunomodulator or pharmaceutical composition according to claim 12 wherein the autoimmune disease or autoimmune disorder further involves inflammation of the muscular layer of a blood vessel or myocardium.

14.

An immunomodulator or pharmaceutical composition according to any one of claims 11 to 13 wherein the autoimmune disease or autoimmune disorder is one that is preceded or caused by a vascular disorder.

fifteen.

An immunomodulator or pharmaceutical composition according to any one of claims 11 to 14 wherein the autoimmune disease or autoimmune disorder is selected from one or more of the following: arthritis, particularly rheumatoid arthritis, psoriasis, psoriatic arthropathy, scleroderma , thyroiditis, intimal hyperplasia after transplantation, graft rejection and vascular disorders, in which the vascular disorder is a vascular disorder that comprises an autoimmune element.

16.

An immunomodulator or pharmaceutical composition according to claim 15, wherein the vascular disorder is selected from one or more of the following: Raynaud's disease or phenomenon, anterior uveitis, obliterating vascular disorder, atheroma formation (also known as arteriosclerosis ), arteritis, myointimal hyperplasia (natural or after angioplasty), inflammatory and autoimmune thickening of the intima and / or muscular layer of blood vessels, lesions of inflammatory blood vessels, atherosclerotic heart disease, reperfusion injury, alterations of cardiac conduction, myocarditis, myocardial infarction.

17.

An immunomodulator or pharmaceutical composition according to claim 15 wherein the graft rejection is chronic graft rejection.

18.

An immunomodulator or pharmaceutical composition according to claim 15 wherein the graft rejection is chronic graft rejection in the absence of immunosuppressant.

19.

An immunomodulator or pharmaceutical composition according to any one of claims 11-18 wherein said bacterium is selected from the group consisting of Tsukamurella paurometabola and Tsukamurella inchonensis.

twenty.

An immunomodulator or pharmaceutical composition according to any one of claims 11 to 19 wherein said bacterium is dead.